PROJECT SUMMARY The research goal of this proposal is to understand the genetic variation, evolution, and molecular functions of primary body axis determinants in dipteran insects (midges/mosquitoes and other flies). A new and widely applicable transcriptome-profiling approach was used in preliminary studies to identify structurally unrelated genes and transcript variants that specify head development and embryo polarity in midges (Clogmia albipunctata, Chironomus ripraius), even though their embryos are otherwise genetically and morphologically similar to other flies. The genetic variability of these anterior determinants across species raises questions about evolutionary constraints in fundamental developmental processes of complex organisms and about the developmental consequences when these long-range pattern organizers are substituted. The project combines functional and comparative approaches to examine the mechanisms of action of anterior determinants in dipterans. The first specific aim of this proposal is to improve genetic resources for the prioritized new model organism Clogmia albipunctata, and to examine the evolutionary context in which its newly identified anterior determinant (encoded by Cal-opaC) evolved. This will be done using transcriptome-profiling in related dipteran species, such as sand flies and mosquitoes. The second specific aim is to characterize in vivo the mechanism of action of Cal-opaC and to compare the mechanism of AP axis specification in Clogmia to the respective mechanisms in other lower dipterans, including the midge Chironomus riparius and the soldier fly Hermetia illucens. The outcome will help to understand why key components of some fundamental developmental pathways are more vulnerable to evolutionary change than others and can potentially reveal non-canonical modulation strategies of fundamental signaling pathways that are important for targeted therapeutics in biomedicine.